Octameric mitochondrial creatine kinase induces and stabilizes contact sites between the inner and outer membrane

Abstract

We have investigated the role of the protein ubiquitous mitochondrial creatine kinase (uMtCK) in the formation and stabilization of inner and outer membrane contact sites. Using liver mitochondria isolated from transgenic mice, which, unlike control animals, express uMtCK in the liver, we found that the enzyme was associated with the mitochondrial membranes and, in addition, was located in membrane-coated matrix inclusions. In mitochondria isolated from uMtCK transgenic mice, the number of contact sites increased 3-fold compared with that observed in control mitochondria. Furthermore, uMtCK-containing mitochondria were more resistant to detergent-induced lysis than wild-type mitochondria. We conclude that octameric uMtCK induces the formation of mitochondrial contact sites, leading to membrane cross-linking and to an increased stability of the mitochondrial membrane architecture.

Figure 1. Immunoblot analysis of uMtCK expression in mouse tissues

Protein extracts of WT and uMtCK-expressing (+/+) mice were separated by SDS/PAGE and immunoblotted. (A) Protein extracts from liver, heart and brain, blotted with an anti-MtCK-antibody. (B) Protein extracts from liver (total), liver cytoplasm (cytopl) and liver mitochondria (mito), blotted first with an anti-MtCK antibody, followed by an anti-VDAC antibody. The results from two WT and two uMtCK-expressing animals are shown.

Figure 2. Ultrastructure of transgenic and WT mouse liver tissue

Liver tissue samples were prepared for transmission electron microscopy as described in the Experimental section: transgenic liver (A), WT liver (B), transgenic liver immuno-reacted with anti-MtCK antibodies (C), transgenic liver immunoreacted with pre-immunization serum (D), MIM/MOM contact sites (indicated by arrows) (E). Scale bar, 200 nm. (F) The number of contact sites was divided by the mitochondrial circumference, calculated from 20 randomly chosen mitochondrial sections in each group. The difference in the number of contact sites between transgenic and WT mice was statistically significant (P<0.0005 as assessed by ANOVA). Results are means±S.E.M.

Figure 3. Detergent-resistance of uMtCK-containing mitochondria

Mitochondria were suspended in sucrose-based medium at a concentration of 0.5 mg of protein/ml. Detergent was added and the turbidity of the mitochondrial suspension was monitored at 540 nm. In parallel, aliquots of the mitochondrial suspension were taken from the cuvette after 30 min and stained with 2% ammonium molybdate for electron microscopy. (A) Rate of change in turbidity as a function of digitonin concentration for uMtCK-containing mitochondria (grey diamonds) and WT mitochondria (filled circles). Inset shows turbidity for: WT mitochondria plus digitonin, 0.5 mg/mg mitochondrial protein (trace d); uMtCK-containing mitochondria plus digitonin, 0.5 mg/mg mitochondrial protein (trace c); WT mitochondria without detergent (trace b); uMtCK-containing mitochondria without detergent (trace a). (B) Rate of change in turbidity as a function of TX-100 concentration for uMtCK-containing mitochondria (grey diamonds) and WT mitochondria (filled circles). Results in (A and B) are the means±S.E.M. of three different mitochondrial preparations. Points marked with asterisks were statistically different for uMtCK-containing mitochondria compared with the control (P<0.001 by ANOVA). (C) uMtCK-containing mitochondria without detergent. (D) WT mitochondria without detergent. (E) uMtCK-containing mitochondria plus digitonin, 0.5 mg/mg mitochondrial protein. (F) WT mitochondria plus digitonin, 0.5 mg/mg mitochondrial protein, in negative staining. Scale bar in (C–D), 1 μm.

Figure 4. Effects of TSAC and doxorubicin on detergent-resistance of uMtCK-containing mitochondria

Turbidity measurements were performed as described in Figure 3. Mitochondria from WT and uMtCK-expressing mice were incubated in measurement medium supplemented with either TSAC (A) or 50 μM doxorubicin (B). Digitonin (0.5 mg/mg of mitochondrial protein) was added, and the rate of change in turbidity was calculated as described in the Experimental section. Results are the means±S.E.M. for three different mitochondrial preparations.